ES2589014T3 - Off-axis interferometer - Google Patents

Abstract

Off-axis digital holographic microscope, comprising: - a partially coherent light source spatially and temporarily, arranged to produce a first partially coherent beam of light (1); - a registration plan (10); - a microscope objective (ML1); - an object cell (Sa) arranged to maintain a specimen to be studied in a frontal focal plane of said microscope objective, said object cell (Sa) being optically conjugated with said registration plane (10); - a grid (G) located in a plane optically conjugated with said registration plane (10), said grid (G) defining a first and a second optical path, and said grid (G) being arranged to divide the first beam of light in a diffracted beam of light of a non-zero order along the first optical path as a reference beam of light, and a second beam of light with a different order of diffraction along the second optical path; - a first lens (L5) located at a point posterior to the objective of the microscope, said grid (G) being located in the posterior focal plane of said first lens; - a second lens (L6), the grid (G) being located in the frontal focal plane of said second lens (L6) and a third lens (L7) optically coupled to said second lens (L6), said registration plane ( 10) located in the posterior focal plane of said third lens (L7) and said third lens being arranged to recombine the reference light beam and the second light beam to obtain a recombined beam of light that will affect the registration plane, in which they interfere with each other, in which the optical path of the reference light beam and the optical path of the second beam of light do not differ by an amount greater than the coherence length of the light source.

Description

DESCRIPTION

Off-axis interferometer

5

Field of the Invention

[0001] The present invention relates to an off-axis digital holographic microscope.

10 State of the art

[0002] In interferometers of the prior art, an incident light beam is usually divided into a beam of the object and a reference beam that are then recombined in a registration plane in which the object beam and the reference beam interfere and produce interference bands. The purpose of such devices is the measurement of the

15 complex amplitude of light (i.e. phase and amplitude information).

[0003] In general, the light used in such measurement has a high coherence, such as the light produced by lasers. This has several drawbacks, such as the appearance of coherent noise (speckled field or speckle) and the high cost related to high coherence light sources.

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[0004] In many cases, a small angle is introduced between the object beam and the reference beam in order to obtain spatially low frequency heterodine bands, as described in US7002691. In general, these types of configurations are called off-axis configurations, due to the non-zero angle between the axis of the interferometer and one of the interfering beams.

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[0005] In such an off-axis configuration, it is essential to have a high coherence incident light to observe interference: if the differences between the path length of the reference beam and that of the object beam are greater than the coherence length of the incident beam, no interference can be observed and the phase information is lost.

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[0006] This means that, for a partially coherent light temporarily, the difference in the length of the path at different positions of the registration plane introduced by the small angle may suffice to disturb the coherence, so that interference is only observed in a part of the registration plane, in which coherence is maintained.

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[0007] The recording of phase and amplitude information (or complex amplitude) forms the basis of the holograph in general and, more specifically, of the digital holographic microscope (MHD). In the MHD, a hologram is registered with a CCD camera and a computer performs the reconstruction of a three-dimensional model of the observed sample. The hologram is obtained by an interferometer. This procedure provides an efficient tool for

40 refocus, cut by cut, depth images of thick samples. MHD allows obtaining quantitative phase contrast imaging with numerous applications such as the observation of biological samples. The depth reconstruction capability makes the MHD a powerful tool for the application of the 3D velocimetrla. Since the holograph provides complex amplitude, powerful processing procedures have been applied, such as automated refocusing, aberration compensation, 3D pattern recognition, segmentation and edge processing.

[0008] The principle of digital holography, with the separation of the object beam and the reference beam, consists in extracting the complex information of an object beam from the interference patterns recorded between the object beam and a reference beam. The complex amplitude can be further processed to calculate

50 digital refocusing and to carry out the formation of quantitative phase contrast images. There are two main types of configuration: online configuration and off-axis configurations.

[0009] The complex amplitude is generally obtained by an interferometer, such as an interferometer of the Mach-Zehnder type or of the Michelson type.

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[0010] In online configurations, as described by I. Yamaguchi et al. in «Phase-shifting digital holography», Opt. Lett. 22, 1268-1270 (1997), the angle between the reference beam and the object beam that affect the camera sensor is the smallest possible. The calculation of the complex amplitude requires a phase stepping procedure, in which several interferometric images are recorded with small changes in the

optical path introduced between the object beam and the reference beam. The optical phase information is calculated by applying the interferometric images in a formula.

[0011] The main disadvantage of the online configuration lies in the need to sequentially register 5 several interferometric images, which limits the acquisition speed due to the frequency of capture of the

camera. In fact, the object must remain static during the entire acquisition operation, the time it takes to register several tables.

[0012] In the off-axis configuration, as described in US 6,525,821 and in "Fourier-10 transform method of fringe-pattern analysis for computer-based topography and interferometry", by Takeda et al, J.

Opt. Soc. Am. 72, 156-160 (1982), there is a non-zero average angle between the object beam and the reference beam that allows the calculation of complex amplitude from a single recorded interferometric image. Regarding the online configuration, this is a decisive advantage for the analysis of phenomena that vary rapidly. However, the use of a Mach-Zehnder or Michaelson type interferometer in these configurations requires an optical source of high temporal coherence. Otherwise, the modulation of stripes is not constant due to the variable optical delays along the field of vision between the object beam and the reference beam.

[0013] As described by Dubois et al. in "Improved three-dimensional imaging with digital holography microscope using a partial spatial coherent source", Appl. Opt. 38, 7085-7094 (1999), and in "Application of digital

20 holographic microscopes with partially spatial coherence sources »in J. of Phys .: Conf. Series 139 (2008), the use of partially coherent lighting improves the quality of the holographic record by decreasing coherent artifacts and noises. In transmission, the most effective noise reduction is obtained by spatially coherent partially illumination. This type of illumination is obtained by reducing the coherence properties of a laser beam or by increasing the spatial coherence of an incoherent source, such as a light emitting diode (LED), by means of an optical filtering system.

[0014] With the arrangement that is commonly used to reduce the spatial coherence of a laser, the laser beam is focused near a tarnished glass in motion. For a given position of the tarnished glass, the light transmitted through the sample constitutes a mottled field. When the tarnished glass is moving,

30 and assuming that the exposure time is long enough to obtain an averaging effect, it can be shown that this type of source is equivalent to a spatially coherent light source whose spatial coherence distance is equal to the average mottled field. This procedure for preparing the source maintains a high degree of temporal coherence that enables the off-axis procedure. However, when short exposure times are required, fluctuations in illumination arise. In practice, it is difficult to make a tarnished glass move fast enough to register a dynamic object that requires a short exposure time.

[0015] With the provision that increases the spatial coherence of an incoherent source or non-laser source, the properties of temporal incoherence are maintained. In this case, the off-axis configuration cannot be applied or

40 record all complex amplitude information in a single box.

[0016] With regard to the placement of samples, two types of main configurations can be defined: differential configuration, as described in EP 1631788, in which the sample is located in front of the interferometer, and the configuration classic defined in EP 1399730, in which the

45 sample is located in an interferometer arm.

Objectives of the invention

[0017] The present invention aims to provide an interferometer that overcomes the disadvantages of interferometers of the prior art.

[0018] More specifically, the present invention aims to provide an off-axis interferometer capable of working with partially coherent light sources.

[0019] In US3572934, a longitudinally inverted shearing interferometer is described, in which a grid is used to divide a coherent beam of light into two interfering beams. The light source used is consistent both temporally and spatially (laser).

[0020] In the article «Time resolved digital holography: a versatile tool for femtosecond laser induced damage

studies », (Laser induced damage in Optical materials, 2009, Proc of SPIE vol. 7504), by A. Melninkaitis et al., describes a holographic recording device comprising a grid for obtaining off-axis interference. The arrangement uses a light source with high spatial coherence and will not be able to register homogeneous interference patterns with a spatially coherent light source. In the article “Resolution- 5 enhanced approaches in digital holography”, (Optical Measurement Systems for Industrial Inspection VI, Proc. Of SPIE, vol. 7389), by M. Paturzo et al., A procedure using a grid of dynamic diffraction phase to synthetically increase the opening of a digital holographic image formation system in a lensless configuration.

[0021] The present invention also aims to provide digital holographic microscope (MHD) configurations that allow off-axis configurations to be used with a source with temporal and / or spatial coherence. In this way the ability to carry out a rapid digital color holographic recording with very low noise levels is achieved.

[0022] The present invention also aims to provide digital holographic microscopes that allow the use of sources with partial coherence created from a source inconsistent with the off-axis configuration. It constitutes a considerable improvement, since it allows the microscope to be operated quickly without the disadvantage of fluctuations due to the configuration with a laser (coherent noise). In addition, this application allows you to use low-cost sources, such as LED, and offers the possibility of simultaneously recording 20 holograms in red-green-blue to provide a fully colored digital holographic microscope without coherent noise.

Summary of the invention

[0023] A first aspect of the invention relates to an off-axis digital holographic microscope as set forth in independent claim 1.

[0024] The expression "off-axis" means that at least one of the interfering light beams has a non-zero angle with respect to the axis of the interferometer, or equivalently, that the interfering light beams are

30 beams not parallel.

[0025] The expression "two optically conjugated planes in an optical system" means that one of the planes is the optical image of the other.

[0026] A grid with a periodicity d divides an incident beam into several beams complying with the condition:

d {without 6m + without fy) = ml

where 0m is the angle between the diffracted light beam and the normal one of the grid, 0i is the angle between the incident light beam 40 and the normal one of the grid, A is the wavelength of light and m is a integer called "diffraction order". The light corresponding to the direct transmission (or specular reflection in the case of a reflection grid) is called "zero order" and is indicated as "m = 0". The other beams of light form angles that are represented by whole numbers m other than zero. Note that m can be a positive or negative value, which results in diffracted orders on both sides of the zero order beam.

Four. Five

[0027] In the attached independent claims 3 and 4, other configurations of the off-axis digital holographic microscope according to the invention are set forth. Other aspects of the invention are set forth in the attached dependent claims.

50 Brief description of the drawings

[0028]

Fig. 1 illustrates the limitation of coherence between two interfering drying beams in a plane.

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Fig. 2 represents an interferometer according to the present invention.

Fig. 3 represents a digital holographic transmission microscope that works in differential mode and comprises an interferometer according to the present invention.

Fig. 4 represents a digital holographic transmission microscope comprising an interferometer according to the present invention.

5 Fig. 5 represents a digital holographic reflection microscope comprising an interferometer according to the present invention.

Fig. 6 represents a digital holographic transmission microscope that works in differential mode and comprises an interferometer according to the present invention.

10

Fig. 7 represents a digital holographic transmission microscope capable of working with fluorescence, comprising an interferometer according to the present invention.

References of the figures

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[0029]

1. First beam of light

2. Second beam of light

3. Third beam of light

20 4. Registration means

5. Beam of diffracted light (non-zero order diffraction)

6. Beam of diffracted parallel light of order other than zero 6 '. Parallel beam of diffracted zero order

7. Light beam not diffracted (or zero order diffraction)

25 8. Optical stop element

9. Beam of incident light

10. Registration plane

11. Compensation means for compensation cradle

12. 2 * Consistency Length

30 13. Area of interference

14. Coherence plane

15. Interferometer

16. Reference mirror

17. Fluorescence excitation light source

35 Bs1, Bs2, Bs3 and Bs4: Beam splitters

EF: Excitation filter G: Grid

L1, L2, L3, l4, L5, L6 and L7: M11 and M12 lenses: microscope objectives 40 M1, M2 and M3: mirrors

P: micro-hole on a screen Sa: Sample or transmission sample holder So: Lighting source RA: Rotating mechanism

45 Rs: Sample or sample holder

W: Cot

[0030] In interferometrla, when partially coherent light is used, it is crucial to maintain the coherence of the incident light in a recording plane to observe interference bands. In many cases, the strips of

50 interference is obtained by dividing a first beam of light (incident) to generate a second and a third beam of light, and recombining the third and second beam of light with a small angle inserted between them.

[0031] In that case, the small coherence length of the partially coherent incident light (the light to be analyzed) introduces a severe limitation: as they are not parallel, the coherence planes of both beams only

55 may interfere in a small area at the intersection between both planes, without any interference being observed when the distance between the coherence planes is greater than the coherence length.

[0032] This is illustrated in fig. 1, in which a beam of light perpendicular to a plane interferes with another beam of light not parallel with an off-axis relative to the plane. As represented, the non-parallel light is only coherent

(capable of interfering) in the proximity of the coherence plane 14, at a distance smaller than the coherence length 12, which defines a limited area 13 in which interference is observed.

[0033] The present invention describes an interferometer in which the coherence plane of an interfering light beam 5 is not perpendicular to the direction of propagation of the light beam in the vicinity of a registration plane.

This provides the ability of the non-perpendicular beam of light to interfere with a beam of perpendicular light and produce a contrast of stripes that does not depend on the position in the registration plane. This allows the registration of off-axis interfering bands (spatially heterodyne bands) even if the light has a limited coherence length, as is the case of the light produced by an LED, a gas discharge lamp, etc. .

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[0034] By using a light beam of partial temporal coherence as the first beam of light (incident), the second and third beam of light in interferometers of the prior art can only interfere in the corresponding zones defined by the coherence length. This means that the difference in the length of the optical path and the phase shift due to the optical devices arranged in the optical path between the second and third

15 light beam should continue to be less than the coherence length of the light source.

[0035] Temporal coherence is the measure of the average correlation between the values of a wave in any couple of moments, separated by a time interval t, which characterizes whether a wave can interfere with itself at a different time. The time interval over which the phase or amplitude varies to a considerable extent

20 (therefore, the correlation is reduced to a considerable extent) is defined as coherence time Tc. At t = 0, the degree of coherence is perfect, while it is considerably reduced in the time interval Tc. The coherence length Lc is defined as the distance traveled by the wave at time Tc. The coherence length can be estimated using the formula:

2ln (2) A2

25 lc = ------------ TT

nn A A

where A is the wavelength of light, AA is the spectral amplitude of the source and n is the refractive index of propagation. For a typical LED source, this represents from some wavelengths to a few tens of wavelengths. For example, for an LED with a wavelength of 650 nm and a spectral bandwidth 30 of 15 nm (typical values for commercially available LEDs), the coherence length is approximately 20A. This means that the off-axis reference beam cannot have a phase shift greater than the coherence time, anywhere in the registration plane. It also assumes that the number of stripes induced by the angular difference between the reference beam and the beam of the object cannot exceed 20, which constitutes a severe limitation.

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[0036] The limitation of the coherence time may also be due to the pulse duration of an ultrashort pulse laser. Normally, said pulse laser has a pulse duration of several femtoseconds, whereby the pulse length is limited to some wavelength. In that case, the coherence time is equal to the duration of the impulse. Again, this assumes that the number of stripes induced by the angular difference

40 between the reference beam and the object beam cannot be greater than the number of wavelength representing the pulse length.

[0037] Preferably, in order to avoid this limitation in both cases, the present invention takes advantage of the particular properties of the diffraction gratings to produce an off-axis reference beam without disturbing the

45 temporal coherence of the interfering beams in the registration plane.

[0038] In interferometer 15 of the present invention, a diffraction grating G is located in the posterior focal plane of a lens L5 located in the optical axes of an incident beam of light 9. The grid G causes the division of the beam of incident light 9 to generate a diffracted beam 5 (reference) and a non-diffracted beam of light 7 (which will become

50 in the object beam). Then, a second lens L6 located at a focal distance with respect to the grid G modifies the shape of the diffracted beam and the non-diffracted beam to form beams parallel to the optical axis. L5, L6 and grid G are selected to obtain, behind L6, two spatially separated beams of light, a diffracted beam and an unfractionated beam of light. The non-diffracted beam of light can become the beam of the object or be eliminated by an optical stop element. In the latter case, another object beam can be provided by a larger optical structure, as will be described later. The diffracted beam is subsequently recombined with the object beam and focused by means of an objective lens L7 in a registration plane, with the registration plane located in the posterior focal plane of L7.

[0039] Being the diffracted beam parallel to the optical axis of L7, but not centered on this optical axis, L7 will introduce an off-axis angle into said diffracted beam.

[0040] Another possibility is that the interfering light beams are any pair of diffracted light beams with a different order of diffraction. For example, the diffracted light beam of order +1 can be chosen as the reference light beam and the symmetrical diffracted light beam of order -1 can be chosen as the object beam.

[0041] Preferably, an optical stop element is used to stop all beams of light except the two two diffracted beams selected to interfere with the registration plane.

[0042] It can be shown that, in said configuration, the grid does not disturb the temporal coherence of any beam of diffracted light of a non-zero order. The temporal coherence is related to the optical path traveled. Therefore, it is equivalent to proving that an optical temporal impulse that illuminates the planes

15 posterior focal points of L5 and L4 at a given time will simultaneously illuminate the entire posterior focal plane of L7.

[0043] A grid G, whose transparency is defined by g (x, y) = (1 + sinKx) / 2, is considered to be illuminated by a monochromatic flat wave of amplitude A. Assuming that the pair of lenses L6-L7 constitutes a system

20 afocal (L6-L7 form a type 4f system), the amplitude in the registration plane is given by:

uv (x, y) = Bexpjj! l "V (f $ + / 7)} '(- * + xf - *, y) (1)

in which B is a constant that does not play a relevant role, j = V-l, v is the optical frequency, c is the speed of light in the vacuum and / 6 and f7 are the focal lengths of L6 and L7. When calculating the contribution of a single beam diffracted in the registration plane, equation (1) becomes:

u'v (x, y) = B'exp jj! "v (# $ + # 7)} exp (-jK * + x- (2)

30 in which K = 2n / A, where A is the spatial period of the grid. B'is a constant that does not play a relevant role.

[0044] Illumination by means of a dirac delta-shaped temporal pulse is obtained by carrying out the Fourier transformation of equation (2):

35 u't (x, y) = B'8 jt-1 (# 6 + # 7) & exp (-; K * + x) (2)

in which t is time, equation (3) expresses the fact that a spatially uniform pulse of light in the frontal focal plane of L6 will simultaneously reach all points of the posterior focal plane of L7 without time interval depending on the position (x, y). The exponential part expresses the fact that the beam strikes the plane of the sensor 40 with an angled angle with respect to the optical axis. Therefore, when the optical trajectories of the interferometer of the present invention are equalized, it will be possible to record the interference pattern in the entire sensor plane between the object beam and the reference beam. The inclined angle of the reference beam provides off-axis configuration.

[0045] Because the demonstrated property of said grid does not depend on the beam of light not diffracted, the arrangement described, which comprises a grid G located in a plane conjugated with the registration plane 10, can be used in any arrangement outside shaft This conjugation can be obtained, for example, by a type 4f system with a grid between the two lenses L5, L6 and a focusing lens L7.

[0046] At this stage, it can be seen that the diffracted beam, which now has its coherence plane parallel to the registration plane, can interfere with any light beam with an equivalent optical path and with its coherence plane parallel to the plane register. More specifically, it can interfere with the beam of non-diffracted light, but also with diffracted light beams of another order, or a beam of light that passes through another arm of a Mach-Zehnder type or larger Michaelson type interferometer. size, provided that the difference in the optical path does not exceed the coherence length of the incident light.

[0047] In the off-axis digital holographic microscope (MHD) of the present invention, digital holograms are recorded by partially coherent light sources. To obtain said partially coherent light sources, incoherent light sources such as LEDs can be used. To obtain partial consistency

5 necessary to observe the interference bands that are required to determine the phase information of the incoming light, a spatial filter can be used.

[0048] In fig. 3 shows a first example of a microscope that makes use of an interferometer 15 according to the invention. In this figure, a partially coherent light source So, such as an LED,

10 is located in the posterior focal plane of an L1 lens. Then, the beam of light produced is spatially filtered through a micro-hole P on a screen in order to increase its spatial coherence. The micro-hole P is located in the posterior focal plane of a lens L2, to illuminate the sample Sa. The sample Sa is located in the posterior focal plane of the objective of the ML1 microscope and then follows the interferometer 15 as described above. The hologram in this figure is recorded by means of a CCD camera.

fifteen

[0049] In the latter case, a cradle W is preferably inserted in the optical path of one of the second or third beam of light, in order to cause a slight displacement of the images produced by the diffracted light beams and not diffracted, in order to obtain the differential interferogram as described in EP 1631788. In this case, compensation means 11 are preferably introduced into the

20 optical path of the non-diffracted beam of light to compensate for the phase shift introduced by the cradle in the diffracted beam.

[0050] Another possibility is to introduce the interferometer into a larger optical structure, such as a Mach-Zehnder interferometer represented in the MHDs illustrated in figs. 4 to 7. In that case, the light beam

25 not diffracted 15 is stopped by an optical stop element 8 and the object beam is provided by another optical path, for example another arm of a Mach-Zehnder type interferometer.

[0051] As with the usual holographic microscopes, a first beam of light 1 is divided into a second beam of light 2 and a third beam of light 3 by means of a first beam splitter Bs1, and recombined by

30 a second beam splitter Bs2 to give rise to a recombined beam, and said second and third beam of light interfere with the recombined beam and form an interference pattern in a recording medium, such as a CCD sensor, in order to Get interference patterns. In that case, the interferometer 15 of the present invention is inserted into the reference arm of the Mach-Zehnder type interferometer in order to obtain the off-axis configuration, the non-diffracted beam 6 'being stopped by an optical stop element 8.

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[0052] Preferably, the presence of the L5 and L6 lenses is compensated by the L3 and L4 lenses in the object beam path in order to compensate for the phase modification induced by said L5 and L6 lenses.

[0053] Preferably, the lens L7 is placed after the beam splitter Bs2 of the Mach-40 Zehnder type interferometer, used to recombine the object beam and the reference beam. This allows sharing said L7 lens between the

object and reference beam.

[0054] Another possibility is to replace the L7 lens with two lenses located respectively in the optical path of the reference beam and in the optical path of the object beam, with both lenses focusing on the means

45, but located before recombination means.

[0055] Also configurations that include a source of fluorescence excitation can be incorporated, as shown in fig. 7.

[0056] Because it is not necessary to use any phase shift, the described MHD can be used to record dynamic scenes very quickly, registering several successive frames in order to record 3D rendering time sequences of the sample being To watch.

[0057] This application allows the use of low-cost sources, such as LEDs, and, with color sensors, allows simultaneous registration of red-green-blue holograms by lighting with three LEDs, in order to provide a microscope Full color digital holography without consistent noise. Until now, the application of the digital color holographic microscope requires the use of complex registration procedures. In the described invention, the three colors can be registered simultaneously.

[0058] There are several types of color sensors on the market, including single color sensors, such as triple sensor designs and color CCDs. In triple sensor designs, a prismatic block (that is, a trichroic assembly comprising two dichroic prisms) can filter the interferogram obtained to generate the three primary colors, red, green and blue, directing each color towards a coupling device of load (CCD) or sensor

5 active pixels (CMOS image sensor) mounted on each side of the prism.

[0059] There are several types of transmission grilles that can be applied. The simplest type of grid is the Ronchi grid, which is formed by a transparent optical plate on which parallel opaque lines are printed with a width I. There is a constant transparent space L between the consecutive opaque lines. Grilles

10 Ronchi often have a transparent opening width equal to that of the opaque. The significant amount that characterizes a Ronchi grid and the diffraction angle for a given wavelength is the grid period P = L + 1. The diffraction analysis of a Ronchi grid is carried out by first decomposing the transmittance function according to a Fourier series. For a given wavelength, each Fourier component results in an order of diffraction characterized by a diffraction angle 0m, in which m is an integer 15 and 0 is the first diffraction angle.

[0060] The diffracted amplitude in each diffraction order is proportional to the corresponding Fourier component. In the interferometer of the invention 15, the period of the grid is selected so as to ensure a spatial separation of the diffracted beams in the plane, in which the optical stop 8 is located.

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[0061] Usually, what is maintained is one of the diffraction orders (m = + 1 or -1) for the reference beam that affects the detector. A limitation of the Ronchi grid consists in the dispersion of the intensity of the light between several diffraction orders, which reduces the available light for interferometric measurements.

[0062] To optimize the efficiency of diffraction in the order of diffraction maintained for the holographic procedure, a graded diffraction grating can be applied. The stepped diffraction gratings also have a periodic structure on one of the surfaces of an optical plate. In this case, it is a tooth-shaped relief of a surface that optimizes the efficiency of diffraction in diffraction orders m = 1 or m = -1.

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[0063] To optimize the efficiency of diffraction, it is also possible to incorporate thick phase holographic grids. This type of grid is obtained by recording in a photosensitive material, for example dichromated jelly, the interference pattern between two flat waves. Subsequently, the plate is processed and is able to diffract mostly light in an order of diffraction according to a Bragg diffraction mode. The angle between the wave

35 of the registration plane determines the period of the grid.

[0064] Then, the analog outputs can be digitized and processed by a computer in order to obtain a three-dimensional color representation of the samples.

[0065] A temporary and spatial partial source may consist of a source (So), a collimation lens (L1), a micro-hole (P) and a lens (L2). Temporal coherence is obtained from the spectral width of the source (So). Normally, it can be an LED with a spectrum that has a peak (for example, the wavelength A = 650 nm, AA = 15 nm), or a set of LEDs that provide a set of peaks, to achieve a holographic record in color. The beam is collimated by the lens (L1) and filtered through the micro-hole (P) in order to increase spatial coherence. It can be shown that the dependence of the emerging spatial coherence, which arises from the lens (L2), is uniform and can be modeled by a coherence function y (x1 - X2, y1 - y2), in which (xi, yi ) and (x2, y2) are spatial coordinates perpendicular to the z-axis.

[0066] This application of the partially coherent source does not imply any restriction and can be practiced in different ways, such as with a laser beam de-correlated by means of a tarnished glass

moving.

Description of the preferred embodiments of the invention 55 Mach-Zehnder Configuration

[0067] Fig. 4 illustrates the digital holographic microscope scheme based on a Mach-Zehnder type configuration that allows off-axis registration with optical sources with spatial and temporal partial coherence.

[0068] In the case of fig. 4, the light source is constituted by a source (So), a collimation lens (L1), a micro-hole (P) and a lens (L2).

[0069] After a reflection in the mirror M1, the beam is divided by Bs1 to generate the beam of the object that illuminates

5 the sample in the transmission and the reference beam, which is redirected to the lens of the ML2 microscope. The image of

frontal focal plane of MI1 It is carried out by the set of lenses MI1, L3, L4 and L7. To form the image, the posterior focal plane of L3 corresponds to the frontal focal plane of L4 and the sensor is located in the posterior focal plane of L7.

[0070] In this configuration, the interferometer of the invention 15 is represented by lenses L5, L6 and L7 and grid G.

[0071] The reference arm is provided with elements corresponding to the MI1, L3 and L4 lenses; respectively, the MI2, L5 and L6 lenses, such that the reference beam and the object beam, except for the sample,

15 the grid G and the optical stop element 8 are almost identical. This guarantees a correct alignment of the two beams in the sensor, in which they interfere. The partially spatially coherent nature of the illumination as! it requires.

[0072] The frontal focal plane of the MI2 lens, in which an image of the optional component C is formed, which

20 can be a compensator and / or an attenuator of the optical path, in the posterior focal plane of l5, which also

it is the frontal focal plane of the L6, in which the grid G is located. The function of the grid is to redirect the light by diffraction, so that the object beam and the reference beam that affect L7 are spatially separated and They propagate in parallel. As already shown, this arrangement with L5, G, L6 and L7 allows to maintain the temporal coherence in the sensor plane in the case of partial temporal coherence, and produce an off-axis interference.

[0073] The L7 lens superimposes the object and reference beams on the sensor with an average angle between them that originates from grid diffraction. In this configuration, the mirror M2 is parallel to Bs2, and M3 is parallel to Bs1. The relative orientation of the beam splitters and mirrors allows adjustment, without changing the beam positions

30 in the sensor, the optical path rotating the rotation mechanism RA, to which the mirror M2 and the beam splitter Bs2 are firmly fixed. Therefore, the configuration allows equalizing the beams of optical reference and trajectory of the object.

[0074] This configuration can be adapted to reflective samples, as shown in fig. 5. In that case, the sample holder Sa is removed and the mirror M2 is replaced by a third beam splitter Bs3 that illuminates a

Reflective sample Rs through the lens of the ML1 lens. The light reflected by the sample and focused by the objective lens is then redirected by a third beam splitter Bs3 towards the second beam splitter, in the same way as in the previous configuration. The same modification is applied to the optical path of the reference beam, replacing the sample with a reference mirror 16.

40

Differential configuration

[0075] The Mach-Zehnder configuration is suitable for applications where the variations in the optical path introduced by the object are limited: in the case of a very fluctuating object thickness, the density

45 of the stripes may end up being too high to be registered by the sensor.

[0076] Furthermore, with a reduced temporary partial illumination, in the Mach-Zehnder configuration, fine tuning of the optical path when the object is changed can be difficult. For this reason, the differential digital holographic microscope was proposed. In the latter case, what is measured is the differential optical phase, which has the advantages

50 of an increased dynamic range for phase measurement and permanent adjustment of the interferometer, regardless of the thickness of the sample.

[0077] The off-axis configuration with partially coherent sources, spatially and temporarily, can be advantageously used in differential mode. The optical scheme is represented by fig. 6.

55

[0078] The partially spatially and temporally coherent source is a source (So), a collimation lens (L1), a micro-hole (P) and a lens (L2). The beam is collimated by the lens (L1) and filtered by the micro-hole (P) to increase spatial coherence.

[0079] After a reflection in the mirror M1, the light beam illuminates the sample and is transmitted by the microscope lens MI1. The beam of light from MI1 is divided by Bs1 into two beams corresponding to a second and a third beam of light. The image of the frontal focal plane of MI1 located within the sample is formed by the set of lenses MI1, L3, L4 and L7. To that end, the posterior focal plane of L3 coincides with the focal plane

5 front of L4 and the sensor is located in the back focal plane of L7. Similarly, the image of the frontal focal plane of MI1 is formed by the set of lenses Ml 1, L5, L6 and L7, the posterior focal plane of L3 corresponding to the frontal focal plane of L4. The distance between L4 and L7 is identical to the distance between L6 and L7.

[0080] Therefore, an image is formed on the CCD sensor of the same plane as the sample plane, 10 corresponding to the frontal focal plane of MI1. A shift between the images formed by the

second and third beam of light by slightly rotating the mirror M2 or the mirror M3. The offset is only a few pixels, or even less than one pixel in the CCD sensor. The elements corresponding to the lenses L3 and L4 of the first optical channel 1, respectively the lenses L5 and L6, are located in the optical path of the third beam of light, such that the optical path of both the second and third beam of light , with the exception of the sample, the grid G and the optical stop element 8, are identical. This allows a correct alignment with the small displacement of the two channels in the sensor in which they are interfering.

[0081] An image of the frontal focal plane of the lens MI1 is formed in the posterior focal plane of L5, which is also the posterior focal plane of L6, in which the grid G is located. The function of the grid consists of redirecting the light

20 by diffraction so that the incident beams of the two channels in L7 are spatially separated. The L7 lens superimposes the two beams on the sensor with a medium angle between them that originates from the grid diffraction. In the configuration, the mirror M2 is almost parallel to Bs2 and M3 to Bs1. These relative orientations of the beam splitters and the mirrors allow adjustment, without changes in the beam positions in the sensor, of the optical path by rotation of the rotation mechanism RA to which the mirror M2 and the beam splitter Bs2 are attached 25 firmly. Therefore, the configuration allows equalizing the reference beam and the path of the object.

[0082] The average angle between the object beam and the reference beam in the sensor provides the off-axis configuration. As already shown, there is a correct alignment, even in the case of a source of partial temporal coherence, in order to homogeneously provide a pattern of stripes contrasted throughout

30 the CCD sensor. It should be noted that an attenuator can be placed in the optical path of the second beam of light to compensate for the loss of light caused by the grid. An optical plate can be inserted in the optical path of the third beam of light in order to compensate for the difference in the optical path introduced between the two channels by the dimmer.

[0083] A fluorescence source 17 can be used when the sample of interest is fluorescent, as shown in fig. 7. Its beam is reflected by the fluorescent beam splitter through the MI1 lens to illuminate the sample. The fluorescent signal that propagates backward is transmitted by MI1 and is spectrally filtered by SF in order to remove the fluorescent excitation part before it hits Bs1. Because the lens MI1 is limited by an aperture, the incoherent fluorescent signal has a partial spatial coherence 40 when it arises from MI1, with the result that the two beams are capable of interfering, provided the displacement is less than that of the spatial coherence length.

Claims (10)

1. Off-axis digital holographic microscope, comprising: 5 - a partially coherent light source spatially and temporarily, arranged to produce a first partially coherent beam of light (1); - a registration plan (10); 10 - a microscope objective (ML1); - an object cell (Sa) arranged to maintain a specimen to be studied in a frontal focal plane of said microscope objective, said object cell (Sa) being optically conjugated with said registration plane (10); fifteen - a grid (G) located in a plane optically conjugated with said registration plane (10), said grid (G) defining a first and a second optical path, and said grid (G) being arranged to divide the first beam of light in a diffracted beam of light of a non-zero order along the first optical path as a reference beam of light, and a second beam of light with a different order of diffraction along the second optical path; twenty - a first lens (L5) located at a point posterior to the objective of the microscope, said grid (G) being located in the posterior focal plane of said first lens; - a second lens (L6), the grid (G) being located in the frontal focal plane of said second lens (L6) and a third lens (L7) optically coupled to said second lens (L6), said registration plane being (10) located in the posterior focal plane of said third lens (L7) and said third lens being arranged to recombine the reference light beam and the second light beam to obtain a recombined beam of light that will affect the registration plane, in which they interfere between yes, in which the optical path of the reference light beam and the optical path of the second light beam do not differ by an amount greater than the coherence length of the light source. 30 2. Digital holographic microscope according to claim 1, wherein the object cell (Sa) is illuminated by the first beam of light (1), said microscope objective (ML1) being located at a point before the grid (G) and in which there is a cradle located in the second optical path to produce a differential hologram. 35 3. Off-axis digital holographic microscope, comprising: - a light source partially coherently spatially and temporarily, arranged to produce a first partially coherent beam of light (1); 40 - a registration plane (10); - a microscope objective (ML1); - an object cell (Sa) arranged to maintain a specimen to be studied in a frontal focal plane of said microscope objective, said object cell (Sa) being optically conjugated with said registration plane (10); - a grid (G) located in a plane optically conjugated with said registration plane (10); 50 - a Mach-Zehnder type interferometer, comprising a first beam splitter (Bs1) and a second beam splitter (Bs2), said first beam splitter (Bs1) being arranged to divide said first beam of light into a second beam of light (2) and a third beam of light (3); - a first lens (L5) located in the optical path of said third beam of light (3), arranged to focus said third beam of light on said grid (G); - a second lens (L6) with the same optical axis as the first lens (L5) and located at a focal distance from the grid (G), arranged to produce at least one beam of diffracted light of order other than zero, said second beam splitter (Bs2) arranged to recombine said second beam of light and said beam of diffracted light to produce a beam recombined; - an optical stop element arranged to stop the zero order diffracted light of said third beam of light; 5 - recording means arranged to record interferometric signals produced by the interaction between the second beam of light and the diffracted beam of light, said recording means being located in the registration plane (10) of said interferometer (15); - focusing means (L7) arranged to focus said recombined beam on said recording means, 10 being the optical path of the second and third light beam essentially equivalent and the cell being located of the object (Sa) and the objective of the microscope in front of the first beam splitter (Bs1), defining a differential holographic configuration. 4. Off-axis digital holographic microscope, comprising: fifteen - a light source partially coherently spatially and temporarily, arranged to produce a first partially coherent beam of light (1); - a registration plan (10); twenty - a microscope objective (ML1); - an object cell (Sa) arranged to maintain a specimen to be studied located in a frontal focal plane of said microscope objective, said object cell (Sa) being optically conjugated with said plane 25 registration (10); - a grid (G) located in a plane optically conjugated with said registration plane (10); - a Mach-Zehnder type interferometer, comprising a first beam splitter (Bs1) and a second beam splitter 30 (Bs2), said first beam splitter (Bs1) being arranged to divide said first beam of light into a second beam of light (2) and a third beam of light (3); - a first lens (L5) located in the optical path of said third beam of light (3), arranged to focus said third beam of light on said grid (G); 35 - a second lens (L6) with the same optical axis as the first lens (L5) and located at a focal distance from the grid (G), arranged to produce at least one beam of diffracted light of order other than zero, said second beam splitter (Bs2) arranged to recombine said second beam of light and said diffracted beam of light to produce a recombined beam; 40 - an optical stop element arranged to stop the zero order diffracted light of said third beam of light; - recording means arranged to record interferometric signals produced by the interaction between the second beam of light and the diffracted beam of light, said recording means being located in the registration plane (10) 45 of said interferometer (15); - focusing means (L7) arranged to focus said recombined beam on said recording means, the optical path of the second and third beam of light being essentially equivalent, and in which the object cell (Sa) and the objective of the Microscope (M11) are located in the optical path of the second beam of light (2). fifty 5. Digital holographic microscope according to claim 4, wherein a second microscope objective (M12) is located in the optical path of the third beam of light (3). 6. Digital holographic microscope according to claim 4, further comprising a third beam splitter (Bs3) located in the path of the second beam of light to illuminate a reflective object (Rs) and a fourth beam splitter (Bs4) located in the path of the third beam of light to illuminate a reference mirror (16), defining a Mach-Zehnder type geometry. 7. Digital holographic microscope according to any of the preceding claims, wherein The partially coherent light source comprises lighting means (So) selected from the group consisting of an LED, a gas discharge lamp, thermal sources and pulse laser. 8. Digital holographic microscope according to any one of claims 3 to 6, wherein said recording means are color-sensitive recording means, and the light source simultaneously produces the minus three different wavelengths, to record a color holographic interferogram. 9. Digital holographic microscope according to claim 7, wherein said light source comprises at least three LEDs of different wavelengths. 10 10. Digital holographic microscope according to claim 7 or 8, wherein the different wavelengths correspond to cyan, magenta and yellow (CMY) or red, green and blue (RGB) for color reconstruction. 11. Digital holographic microscope according to any of the preceding claims, comprising a source of fluorescence excitation (17) optically coupled to said sample holder.